Test elements which have one or more discrete zones containing immobilized reactants are frequently used to detect analytes in a sample or in a sample material. These zone are referred to in the following as analyte detection zones or detection zones. The analyte is bound in a reaction (assay) from the sample in the analyte detection zone by means of a specific interaction with the reactant. The binding events can be detected by measuring instruments e.g. by the optical detection of fluorescence signals. The test elements can be prepared with microarray structures and be present in the form of a biochip for the site-selective detection of binding reactions as described for example in the published applications WO 2007/042219 and EP1412533. The test elements can also be test strips on which for example one or more detection zones containing immobilized reactants are arranged in the form of strips or lines perpendicular to the flow direction of the sample. Such test strips are for example described in WO 2008/105814 and US 2004-0126767.
Labelled binding partners are used to detect the binding events between the reactants and the analyte in the analyte detection zone. These are reactants which comprise a labelling reagent and which interact specifically with the analyte. Fluorophores which emit a light signal after excitation by electromagnetic radiation of a certain wavelength can for example be used as labelling reagents. The measurability of this light signal in the analyte detection zone and its intensity indicate the presence of the analyte in the sample or are a measure for its concentration.
In the case of immunological detection methods the binding events on the test element are based on a specific antigen-antibody interaction (immunoassay). In immunological detection methods according to the sandwich principle the sample to be analyzed is typically brought into contact with a conjugate consisting of a labelling reagent and an analyte-specific binding partner (e.g. an antibody) so that complexes are formed between the analyte—if present in the sample—and the labelled conjugate. These complexes react with the immobilized reactants (e.g. antibodies) on the test element so that sandwich complexes consisting of the analyte-conjugate and the immobilized reactants which can be detected by means of the label, are present in the analyte detection zone. Examples of such immunoassays are described in the U.S. Pat. No. 4,168,146 (Grubb et al.) and U.S. Pat. No. 4,366,241 (Tom et. al.). A competitive assay is an alternative technique. In a competitive assay the labelling reagent is generally conjugated with a binding partner which is identical to the analyte or it is an analyte analogue. In this manner the labelled conjugate competes with the actual analyte for binding to the reactants in the analyte detection zone on the test element. A competitive assay is usually used to detect an antigen if only a single specific antibody is available for this antigen or if the antigen does not have sufficient binding sites for the unhindered binding of two antibodies. Hence, this assay variant is also suitable for detecting haptens. Examples of competitive assays are described in the U.S. Pat. No. 4,235,601 (Deutsch et. al.), U.S. Pat. No. 4,442,204 (Greenquist, et. al.) and U.S. Pat. No. 5,028,535 (Buechler et. al.).
Test elements in the form of immunological test strips are widely used devices for the rapid determination of drugs, pregnancy hormones, infectious diseases or so-called cardiac markers such as troponin T. In this connection qualitative tests which are merely read-out visually and often only give a “yes-no” answer and quantitative tests which are evaluated by means of a readout device are widely used.
Rapid tests for immunologically detectable substances have been known for a long time for a large number of different parameters, for example from WO 97/06439, EP 0 291 194, U.S. Pat. No. 5,591,645, U.S. Pat. No. 4,861,711, U.S. Pat. No. 5,141,850, U.S. Pat. No. 6,506,612, U.S. Pat. No. 5,458,852, U.S. Pat. No. 5,073,484. In these cases the immunological detection reagents (essentially labelled and unlabelled antibodies or antigens) are usually provided in a dry form on a support which allows the transport of a sample liquid (in particular body fluids such as blood, serum, plasma, urine, saliva etc.) on or in the support. For this purpose the support is preferably capillary-active and is for example a membrane or a plastic support provided with capillary channels (such as e.g. in U.S. Pat. No. 5,458,852). Among experts one often speaks about immunological or immunochromatographic test strips or test devices. These terms as well as the term “carrier-bound immunological test” or “carrier-bound immunological test elements” are often used synonymously and should also be used interchangeably in the following.
In addition to one or more analyte detection zones for the detection of one or more analytes, the test elements usually have a further zone in which the reactants are present which specifically bind the labelled binding partner. This zone is referred to as a control zone or indicator zone and serves as a function check for the labelled binding partner by capturing the labelled binding partner without requiring a direct involvement of the analyte. Analyte detection zone(s) and the control zone are usually spatially narrowly limited and arranged clearly separated from one another on the test element. In test elements in the form of a test strip in which the sample is applied in a liquid form and reaches the various zones with the aid of capillary forces, the control zone in the form of a control line is usually downstream of the analyte detection zone on or in the material through which a flow is possible. On such test strips the control zone additionally serves as a run check in order to ensure that the labelled binding partner has also actually reached the individual zones. The function of the control zone as a check for the functionality of the labelled binding partner and as a run check for the functionality of a test strip is also referred to by a person skilled in the art as “control function”.
A test element is described in US 2007/0048807 A1 which has an indicator zone in addition to the detection zone on a porous membrane. A material (receptive material) is immobilized in the indicator zone which specifically binds with binding partners conjugated with labelling reagent which are not complexed with the analyte in the analyte detection zone.
Due to the spatial separation of the analyte detection zone and control zone, spatially resolving optical systems such as camera chips or two-dimensional or three-dimensional photodiode arrays for the analysis of the analyte detection zone and control zone are often present in the measuring device for evaluating the test elements. The signals of the optical systems are then converted by an appropriate evaluation software into concentration values and displayed.
If the labelling reagent of the labelled binding partner is a fluorophore, then in addition to checking the function of the labelled binding partner by means of a control zone, it is also necessary to calibrate the analyte-specific measurement signal. The analyte-specific measurement signal is the signal which can be generated in the analyte detection zone due to the presence of the analyte in the sample. The intensity of this signal depends on the irradiated energy in the excitation spectrum of the fluorophore which is localized by the analyte-specific interaction in the analyte detection zone. Experience shows that the energy generated by the radiation source is in turn subject to variations which, on the one hand, can be attributed to production-related variations between different radiation sources of the same type or, on the other hand, arise over the cycle of use of a radiation source. Such variations have to be corrected for a reproducible and reliable quantitative determination of the analyte. This is usually carried out by calibrating the analyte-specific measurement signal with a signal that has been generated by a standardized amount of the fluorophore. The standardized amount of the fluorophore is also referred to by a person skilled in the art as a fluorophore standard or calibration standard. The radiation emitted by the fluorophore standard after excitation with electromagnetic radiation is usually referred to as the “calibration signal”.
In the systems used in the prior art for the (semi)quantitative determination of analytes, the analyte-specific measurement signal can be calibrated by generating the calibration signal outside the test element (for example TRIAGE® system, Biosite Inc.).
In addition there are systems in which the calibration of the analyte-specific measurement signal can be carried out directly with the test element. Such test elements comprise, in addition to the analyte detection zone and optionally the control zone, a further discrete zone spatially separated from the other zones in which the fluorophore as a labelling reagent is disposed in a defined amount directly on the test element. This zone is referred to in the following as a calibration zone. The presence of the calibration zone on the test element enables the calibration signal to be detected directly on the test element using the same measuring device or analyzer that also detects the signals of the analyte detection zone(s) and/or the control zone. In the case of a test strip or microarray, the signal detection can be carried out using the same spatially resolving optical system as the function check and measurement of the analyte-dependent signal in the analyte detection zone. In immunological test strips the calibration zone can for example be present in the form of a further line perpendicular to the flow direction of the sample (for example CARDIAC READER®, Roche Diagnostics).
The presence of a calibration zone on the test strip has the advantage that the stability requirement for the fluorophore in this zone is reduced to the life time of the test element lot. In contrast, if the calibration signal were to be generated outside the test element, the user would not only have to take care that the test strip is adequately stable but also that the separately used fluorophore standard is stable enough, whereby an additional source of error has to be avoided in each case. Since the generation and detection of the calibration signal outside the test element usually requires additional radiation sources, filters etc. and a separate optical evaluation system, the corresponding analyzers have a more complex construction, are larger and usually also more expensive.
The term “calibration function” is also commonly used in connection with the phrase “calibration of the analyte-specific measurement signal”. The term “calibration function” is known to a person skilled in the art just like the phrase “calibration of the analyte-specific measurement signal” and relates to means that are used to generate the calibration signal within an analyzer for example on a test carrier or, as described above, outside a test carrier.
A test element can also be used to determine several different analytes simultaneously in a sample. In the case of such “panel tests” a discrete detection zone containing analyte-specific reactants is present for each analyte on the test element. Since one analyzer is usually used to evaluate different test elements for determining different medical parameters in the laboratory or by practicing doctors, the corresponding test elements must have a uniform size which is standardized with respect to the instrument. For this reason, less and less space is available for the control and calibration zone as the number of analyte detection zones on the test element increases. Moreover, the size of the test element itself is limited by the requirement of an adequate wetting of the test area by the usually small sample volumes. Since in addition there must be a spatial separation between the calibration and analyte detection zone, the number of analyte detection zones on the test element is limited and thus also the number of analytes that can be detected using one test element. In order to nevertheless detect as many analytes as possible with the aid of a test carrier for a panel test, it is therefore often necessary to carry out the measurements required to calibrate the analyte-specific measurement signal outside of the test element. However, this requires correspondingly complicated adaptations of the analyzer or measuring instrument and/or the use of additional test elements and, for the above-mentioned reasons, also requires more time and effort by the laboratory staff.
It is against the above background that the present invention provides certain unobvious advantages and advancements over the prior art. In particular, the inventor has recognized a need for improvements in a test element having a combined control and calibration zone.